Protozoan parasites are single-celled, eukaryotic pathogens that cause among the most deadly and widespread diseases today. Combating this global scourge requires a more comprehensive understanding of parasite biology, as well as identifying and developing new targets for therapeutic intervention. Protozoan parasites deploy transmembrane adhesin proteins to mediate close and strong contact with the host cell for the purpose of invasion. Disease follows from successful invasion, making interfering with the invasion program an attractive therapeutic strategy. The adhesive contacts between parasite and host must ultimately be dismantled for parasite internalization and sealing of the host membrane. At the heart of this process are parasite rhomboid proteases that catalyze the essential cleavage of adhesin proteins. Rhomboid proteases are unusual membrane proteins with a serine protease active site assembled within the membrane. Biochemical complexity of these extraordinary enzymes has long presented obstacles to investigating their mechanism of action. We developed a pure enzyme reconstitution system for studying rhomboid catalysis directly, and identified the role for one protozoan rhomboid in host-cell invasion. Through these advances, we have recently built up a detailed structural and functional framework for understanding rhomboid catalysis, as well as developed methods to study malaria rhomboid enzymes directly. Capitalizing on these new advances, we aim to use our most informative engineered variants to delineate the enzymatic reaction, to investigate rhomboid protease conformation directly in membranes, and to decipher how protozoan rhomboid enzymes target their substrates. These insights, in turn, will be applied to the roles of parasite rhomboid enzymes in disease. PUBLIC HEALTH RELEVANCE: Single-celled parasites cause many diseases including malaria, killing millions of people worldwide, and neurologic birth defects and death of AIDS patients within the US. These parasites must get inside human cells to cause disease, and in the past we discovered an unusual class of enzymes involved in cell entry. We are using innovative techniques to study how these enzymes function, and applying this knowledge to combating their roles in disease.